Method of and an apparatus for image analyses of biological specimens

• US 4,741,043 A
• Filed: 11/04/1985
• Issued: 04/26/1988
• Est. Priority Date: 11/04/1985
• Status: Expired due to Term

• Current Assignee: Cell Analysis Systems, Inc. (Becton, Dickinson & Co)
• Original Assignee: Cell Analysis Systems, Inc. (Becton, Dickinson & Co)
• Inventors: Bacus, James W.
• Primary Examiner(s): Boudreau, Leo H.
• Application Number:
  US06/794,937
• Time in Patent Office: 904 Days
• Field of Search: 382/6, 356/39-40, 377/10, 364/416, 364/413, 358/107, 358/93, 350/534, 350/535, 350/536, 350/507, 350/529, 350/531, 206/456, 424/3, 427/2, 128/633
• US Class Current: 382/129
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/127 Calibration; base line adjustment; drift compensation
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts
• CPC Class Codes: G06V 20/693 Acquisition

Abstract

A user interactive system for dynamically testing and evaluating various cells, antigens, or other materials taken from the human body. More specifically, the DNA in specimen cells is analyzed and quantified by image analysis using pattern recognition techniques. The user is provided with a unique slide or support on which there are specimen and reference materials or objects which are simultaneously stained or otherwise image enhanced at the time of analysis.

Claims

1. A method of analyzing cell objects on a support in automatic analyzing apparatus comprising the steps of:

providing a support means which has previously prepared control calibration material thereon,

adding specimen cell objects to the support means adjacent the calibation material,

treating the speciment cell objects and the calibration material at the same time with an image enhancing material,

calibrating the automatic cell object analyzing apparatus by analyzing the treated calibration material on the support means with light and imaging apparatus and adjusting the apparatus based on the analysis, and

measuring and analyzing the specimen cell objects on the support means.

2. A method in accordance with claim 1 in which the calibration material is an optical density reference material and in which the calibrating step includes adjusting the optics of the apparatus relative to a reference material of known optical density.

3. A method in accordance with claim 2 including the step of using the calibration material also as a reference location mark to simultaneously provide a reference location for the support means at the time of adjusting the optics.

4. A method in accordance with claim 1 in which the calibration material comprises control cell objects and in which specimen cell objects are cells and in which the step of treating comprises staining the control cell objects and specimen cell objects at the same time.

5. A method in accordance with claim 4 in which the staining step comprises staining DNA in the cells.

6. A method in accordance with claim 5 including the step of staining DNA within the nucleus of specimen and control cells.

7. A method in accordance with claim 1 in which the calibration material comprises control cell objects and including the step of binding the same monoclonial antibodies to the specimen and control cell objects.

8. A method in accordance with claim 7 including the further step treating the control and speciment cells object with an image enhancing material.

9. A method in accordance with claim 8 in which the monoclonial anitbody is conjugated with an eynzme and wherein the treating step comprises applying thereto a substance with which the enzyme reacts to provide image enhancement.

10. A method in accordance with claim 9 wherein the monoclonial antibody binds to estrogen receptors in the specimen and control cell objects.

11. A method in accordance with claim 8 in which the monoclonial antibody is conjugated with a florescent material.

12. A method in accordance with claim 11 including the step of exciting the fluorescent material on the cell objects at a wave length which excites the fluorescence of said material and analyzing by the step of observing at another wave length at which fluorescent emission occurs.

13. A method in accordance with claim 1 in which the analysis is for a particular virus and in which the control calibration material and specimen cell objects are treated with a nucleic acid probe specific for the genome of said virus.

14. An apparatus for automatically analyzing cells of a specimen as set forth in claim 1 wherein said means for analyzing includes:

means for determining the quantity of DNA contained in a specimen cell.

15. An apparatus for automatically analyzing cells of a specimen as set forth in claim 14 wherein said means for analyzing further includes:

means for determining the number of cells of the specimen having a relative DNA content.

16. An apparatus for automatically analyzing cells of a specimen as set forth in claim 15 wherein said means for analyzing further includes:

means for displaying the distribution of cells of the specimen to indicate the number of cells having a relative DNA content over a range of DNA content values.

17. A cell analysis method for determining the mass of a predetermined material from the optical density of specimen cell objects, said method comprising the steps of:

providing a support with a reference area and a specimen area, the reference area already having a plurality of known refrence cell objects having a known mass of said predetermined material in said reference area;

adding specimen cell objects of an unknown mass of the predetermined material to said support at said specimen area;

measuring the optical density of the reference cell objects;

determining a density factor from said measured reference cell objects and said predetermined stored known mass of said predetermined material of said reference cell objects;

measuring the optical density of said specimen cell objects; and

determining the true mass of said predetermined material of said specimen cell objects from said measured optical density of said specimen cell objects and said density factor.

18. A cell analysis method as set forth in claim 17 further comprising the step of:

determining a phyical characteristic of said specimen cell objects from said determined true mass of said predetermined material.

19. A cell analysis method as set forth in claim 18 wherein:

said reference cell objects and specimen cell objects are red blood cell objects and said determined physical characteristic is mean cell hemoglobin.

20. An apparatus for automatically analyzing cells of a specimen as set forth in claim 17 wherein:

said density factor is a factor indicating the change in optical density of the reference cell objects due to the staining process.

21. An apparatus for automatically analyzing cells of a speciment as set forth in claim 20 wherein:

said stain preferentially stains portions of the reference cell objects and the specimen cell objects.

22. An apparatus for automatically analyzing cell objects of a specimen as set forth in claim 17 wherein said means for calibrating includes:

means for displaying the distribution of light intensity from the calibration area of the slide; and

means for adjusting the light source in a direction such that the displayed distribution substantially matches a reference distribution.

23. An apparatus for automatically analyzing cells of a specimen as set forth in claim 22 where said calibration area is divided into pixels each having a light intensity associated therewith, and said means for displaying includes:

means for displaying a range of different light intensities and the number of pixels of said calibration area which have said different intensities.

24. A microscopic slide for an automated cell analysis system having stored known mass characteristics for cell objects and for use in analysis of specimen cell objects, said slide comprising:

a support having a reference area on one side of said support, said reference area containing reference objects thereon having said predetermined known mass characteristics similar to the specimen objects to be analyzed for a true known mass characteristic at a later time, said reference objects to be image enhanced or analyzed at the time the specimen objects are image enhanced or analyzed for calibrating the apparatus.

25. A microscopic slide as set forth in claim 24 wherein said support further includes:

an identification area having a prefixed optical pattern which can be identified by said apparatus for verifying the integrity of the slide.

26. A microscopic slide as set forth in claim 24 wherein said reference area further includes:

a focusing area having an optical pattern of a predetermined optical density which can be measured and identified by said apparatus.

27. A microscopic slide as set forth in claim 25 wherein:

said optical identification pattern can be identified by measuring the physical dimensions between one or more identifiable features of said pattern.

28. A microscopic slide as set forth in claim 24 wherein:

said reference area is delineated by a pattern which can be detected visually.

29. A microscopic slide as set forth in claim 24 wherein said side further includes:

an identifiable feature which acts as a location reference for the automated cell analysis system.

30. A method of analyzing cell objects on a support useable with an automatic cell analyzing apparatus,

providing a support having control cell objects with known characteristics of a predetermined material,

placing specimen cell objects having an unknown amount of such predetermined material on the support,

putting the support in the apparatus for analyzing the cell objects,

performing an integrity check on the support to determine if it is a valid support having the control cell objects of the known characteristics of the predetermined material for calibrating the cell analyzing apparatus,

disabling the apparatus from analysis of the specimen cell objects of the support is not validated,

and enabling analysis of the cell objects for determining the amount of said predetermined material on the support if the support is validated.

31. A method in accordance with claim 30 in which the step of performing the integrity check comprises optical examining the support for a predetermined optical reference.

32. An automated cell analysis system comprising:

a microscope with focusing means;

means for supporting a specimen slide means with specimen cell objects for mounting on said microscope and control cell objects on said slide means having known characteristics of a predetermined material and having at least one physical characteristic distinguishing whether said slide means is authorized for use in the cell analysis system;

said control cell objects having a known mass of said predetermined material in said reference area;

said specimen cell objects having an unknown mass of the predetermined material;

means for measuring the optical density of said predetermined material in said control cell objects;

means for storing a known cell mass for said reference cell objects,

means for determining a density factor from said measured optical density of said predetermined material in said control cell objects and the stored known mass of said predetermined material for said control cell objects;

means for measuring the optical density of said predetermined material in said specimen cell objects; and

means for determining the true mass of said predetermined material of said specimen cell objects from said measured optical density of said specimen cell objects and said density factor.

33. An automated cell analysis system as set forth in claim 32 wherein:

said at least one physical characteristic is an optical pattern identified by analysis of said optical values.

34. An automated cell analysis system as set forth in claim 33 wherein:

said optical pattern is identified by its physical dimensions.

35. An automated cell analysis system as set forth in claim 33 wherein:

said optical pattern is identified by its average optical density.

36. An automated cell analysis system as set forth in claim 33 wherein:

said optical pattern is identified by having a plurality of identifiable features.

37. An automated cell analysis system as set forth in claim 36 wherein:

said optical pattern is identified by the physical dimensions of said identifiable features.

38. An automated cell analysis system as set forth in claim 36 wherein:

said optical pattern is identified by the optical density of said identifiable features

39. An automated cell analysis system as set forth in claim 36 wherein:

said optical pattern is identified by the physical dimensions separating said identifiable features.

40. An automated cell analysis system as set forth in claim 33 wherein:

said at least one physical characteristic is not identifiable without aid of said microscope.

41. An automated cell analysis system as set forth in claim 33 wherein:

said at least one physical characteristic is not identificable without aid of said identifying means.

42. A cell analysis method for determining the optical density of specimen cells, said method comprising the steps of:

providing a slide with a reference area and having on the slide reference cells of a predetermined optical density in said reference area;

adding specimen cells of an unknown optical density to said slide at a said specimen area on said slide;

staining both said reference cells and said specimen cells with the same stain;

measuring the optical density of the stained reference cels;

determining a staining factor from said measured optical density of said stained reference cells and said predetermined optical density of said reference cells;

measuring the optical density of said stained specimen cells; and

determining the optical density of said specimen cells from said measured optical density of said stained specimen cells and said staining factor.

43. A cell analysis method as set forth in claim 42 wherein said step of staining includes the step of:

preferentially staining only certain portions of said reference cells and said specimen cells.

44. A cell analysis method as set forth in claim 43 wherein said step of preferentially staining said reference cells and said specimen cells includes the step of:

preferentially staining the nuclei or contents of the nucleus of said reference cells and said specimen cells.

45. A cell analysis method as set forth in caim 44 wherein:

said specimen cells are nucleated.

46. An appartus for automatically analyzing specimen cells of a specimen side mounted on a microscope illuminated with a light source, said apparatus comprising:

means for analyzing specimen cells and reference material on the specimen slide each having been treated with an image enhancing material;

means for calibrating the light source on the basis of the distribution of light intensity from a calibration area on the slide;

means for analyzing the cells of a specimen on the basis of the distribution of light intensity from a specimen area on the slide; and

means for enabling said analyzing means on the basis of the distribution of light intensity from an identification area on the slide.

47. An apparatus for automatically analyzing cells of a specimen as set forth in claim 46 wherein said reference area contains reference cells which further includes:

means for calculating a reference parameter on the basis of the light intensity from the reference area on the slide.

48. An apparatus for automatically analyzing cells of a specimen as set forth in claim 47 wherein:

said reference parameter is used to calibrate said means for analyzing the cells of a specimen.

49. An apparatus for automatically analyzing cells of a specimen as set forth in claim 48 wherein:

said reference cells and said specimen cells are stained with the same stain.

50. An apparatus for automatically analyzing cells of a specimen as set forth in claim 47 wherein:

said reference parameter is a factor indicating the change in optical density of the reference cells due to the staining process.

51. An apparatus for automatically analyzing cells of a specimen as set forth in claim 50 wherein:

said stain preferentially stains portions of the reference cells and the specimen cells.

52. An apparatus for automatically analyzing cells of a specimen as set forth in claim 46 wherein said means for calibrating includes:

means for displaying the distribution of light intensity from the calibration area of the slide; and

means for adjusting the light source in a direction such that the displayed distribution substantially matches a reference distribution.

53. An apparatus for automatically analyzing cells of a specimen as set forth in claim 50 where said calibration area is divided into pixels each having a light intensity associated therewith, and said means for displaying includes:

means for displaying a range of different light intensities and the number of pixels of said calibration area which have said different intensities.

54. An apparatus for automatically analyzing cells of a specimen as set forth in claim 52 wherein:

said reference distribution is indicative of the number of pixels of said calibration area which should exhibit a particular intensity.

55. An apparatus for automatically analyzing cells of a specimen as set forth in claim 46 further including:

means for moving said slide so that different areas on the slide can be analyzed; and

means for determining the location on the slide being analyzed.

56. An apparatus for automatically analyzing cells of a specimen as set forth in claim 55 wherein said means for determining includes:

means for determining a reference location on said slide in accordance with the light distribution of a location area on said slide.

57. A method of measuring the hemoglobin in specimen blood cells by an optical density measuring apparatus, said method comprising the steps of:

providing a slide with control blood cells having a predetermined hemoglobin value,

providing a slide with control blood cells having a predetermined hemoglobin value,

placing on the slide the specimen blood cells,

calibrating the optical density apparatus to the known hemoglobin value of the control cells,

measuring the optical density of the specimen cells, and

providing an output indicative of the hemoglobin value for the specimen blood cells.

58. A method in accordance with claim 57 including the step of the measuring the hemoglobin value of each of the individual specimen cells, and calculating a mean cell hemoglobin for the specimen cells.